1. Field of the Invention
The present invention concerns an improved test to determine the lipopolysaccharide- and/or lipid-A- and/or glucan-reactivity in body fluids, a suitable test system for this as well as its usage to determine lipopolysaccharide- and/or glucan-reactivity.
By reactivity is understood in the context of this description the activity of lipopolysaccharide and/or lipid-A and/or glucan, particularly beta-1,3-D-glucan, in a biological liquid as for example plasma. Reactive lipopolysaccharide and/or lipid-A and/or glucan is pathophysiologically and/or pharmacologically-toxicologically active and/or free lipopolysaccharide and/or lipid-A and/or glucan.
2. Description of Related Art
Infections with gram-negative bacteria (as Escherichia coli) or with funghi (as Candida albicans) represent a great medical problem since a long time. The germinates can invade into the blood stream or into the meningeal space where they can cause life threatening conditions, as sepsis or meningitis. Therefore these medically so relevant pathogens should be diagnosed as soon as possible.
However, microbiological procedures to grow these germinates are very time consuming. In many cases the diagnosis can be made not until days or weeks. However, in few hours the clinical condition of the patient can worsen in such a way, that in the worst case he dies due to this infection.
Therefore, it was not missing on to try to shorten the time interval to make a diagnosis. One of theses approaches is the detection of lipopolysaccharides (in the following termed “LPS”), of lipid-A or of glucans with the so called Limulus-Assay. LPS, lipid-A and glucans are dangerous for the patient, because their presence in blood can cause a pathologic disseminated intravascular coagulation (DIC).
The cell wall of gram-negative bacteria partly consists of LPS (formely called endotoxin), which is composed of a harmless polysaccharide part and a for many cells dangerous lipid-A part.
The LPS- and/or lipid-A- and/or glucan-reactivity is of high pathophysiological importance: active LPS and/or free LPS, and/or active glucan and/or free glucan reacts for example very intensively with the monocytes of blood or with the endothelium.
The monocytes can thereby be activated very strongly or can be destroyed, and internal cell compartments get in contact with plasma and thrombocytes.
Since monocytes contain DNA and so-called tissue-factor, the coagulation in sepsis is activated immediately. A life-threateningly enhanced disseminated intravascular coagulation results with e.g. multiple thromboses in small vessels, that can result into multi-organ-failure.
With the present invention a procedure is made available, that imitates the pathophysiological interaction, i.e. the reactivity, of LPS, lipid-A, glucan, respectively, with susceptible cells, whereby the cells and/or cell membranes are substituted in vitro by the Limulus-factors. Therefore, one such procedure that detects sensitively and specifically within minutes LPS, lipid-A or glucans, particularly reactive and/or free LPS and/or reactive and/or free lipid-A and/or reactive and/or free glucans, is of great clinical importance.
According to the state of the art LPS is detected with the Limulus-assay [Hurley J. C. Endotoxemia: methods of detection and clinical correlates. Clinical Microbiology Reviews 1995; 8: 268–292]. The principle of this test is that LPS—but also glucans—activate the coagulogen/coagulin system of the Horseshoe-crab (Limulus polyphemus) called the Limulus-system.
The Limulus-system is a defense system of the horseshoe-crab against invading pathological germinates and represents a primitive system of native immunity.
The limulus-system mainly consists of the following factors: factor C, factor B, factor A and coagulogen.
LPS or lipid-A activates factor C, activated factor C activates factor B, activated factor B activates factor A. Activated Factor A converts soluble coagulogen into insolulbe coagulin.
In the Limulus-system for glucans, that are components of funghi, there is also a factor G, that is activated by glucans, particularly beta-1,3-D-glucans, to a serine protease, whereby activated factor A is formed.
In the Limulus-assay LPS and/or lipid-A and/or glucans of the sample act on Limulus amebocyte-factors, in the following called Limulus-factors, and results into changes, that can be detected in different ways. These Limulus-factors are prepared according to the state of the art from the lysate of Limulus-amebocytes, wherefrom the abbreviation LAL comes.
Examples for detection procedures are the LAL-gelation test, the coagulogen-based LAL assay and the chromogenic LAL assay (see Hurley J. C., Clinical Microbiology Reviews 1995; 8: 272).
In the chromogenic LAL assay or the chromogenic Limulus-test the reaction of the activated Factor A on coagulogen is imitated by substitution of coagulogen by a chromogenic substrate, e.g. S-2834® (Isoleucyl-Glutamyl-Glycyl-Arginyl-paranitro-Anilide=Ile-Glu-Gly-Arg-pNA) or S-2423® (CH3-CO-Ile-Glu-Gly-Arg-pNA) (Chromogenix, Mölndal, Sweden).
The chromogenic Limulus-test is commercially available, e.g. under the trade names Limulus Amebocyte Lysate Endochrome® (Charles River Endosafe; Charleston, USA), Coatest® Plasma Endotoxin or Coamatic® Endotoxin (Chromogenix, Mölndal, Sweden) or Pyrochrome® (Cape Code, Falmouth, USA).
Usually the chromogenic Limulus-test is performed in the so-called end Hyperbolic-Rate-Assay-Kinetic (at 37° C., incubation time=41 min for plasma, product information of Pyrochrome®, Haemochrom, Essen, Germany) i.e. chromogenic substrate and the Limulus-Factors C, B, A are added together to the sample to be tested for endotoxin. Consequently, the activation of the factors C, B, A and the cleavage of the substrate happens at the same time. The longer the assay incubation time is prior to addition of the stop reagent, the more activated factor A is generated and the more chromogenic substrate is cleaved.
Alternatively, the chromogenic substrate can be added not until a time interval after addition of the factors C, B, A. The disadvantage of this is that during the action of the endotoxin on the factors C, B, and A increasingly autocatalytic and/or autodestructive interactions happen (s. FIG. 21). The chromogenic substrate (as added in the Hyperbolic-Rate-Assay) inhibits partially these interactions.
The main-problem with the Limulus-test is, that anti-Limulus factors of the sample, as for example proteins, especially serine protease-inhibitors, as antithrombin III and/or heparin cofaktor II and/or alpha2-antiplasmin, disturb the activation of the Limulus-system. This means that the result of the Limulus-test depends among other things on the content of anti-Limulus factors in the sample, as e.g. proteins, especially serine protease-inhibitors as antithrombin III and/or heparin cofaktor II and/or alpha2-antiplasmin (see FIGS. 12, 14).
This interference is eliminated according to the state of the art by 10 fold dilution of the sample and by heating the diluted sample for 15–20 min at 75° C. prior to the Limulus-test. Optionally the sample may be strong acidified for inactivating anti-Limulus factors (see Obayashi, T. et al. Clin. Chim. Acta 149, 55–65, 1985; J. Lab. Clin Med. 4, 321–330, 1984; U.S. Pat. No. 4,495,294). However, this procedure is problematic, because LPS and/or lipid-A and/or glucans that had been bound to physiologic inhibitors and is hence inactive
can be released from the binding, i.e. the method does not mainly measure reactive and/or active and/or free but also bound LPS or lipid-A or glucan, respectively, with the consequence that this method is not routine-suited. Inhibited LPS, lipid-A, or glucan, respectively, has subordinate pathophyiological importance. Additionly, the active or free LPS or lipid-A molecules of the sample are nearly completely inactivated by heat treatment. The commercially available Limulus-tests have not found entrance into the clinical routine among other things due to these problems.
For example there are plasmatic antibodies and other binding-proteins, as the Lipopolysaccharide-Binding-Protein (LBP), that neutralize LPS and/or lipid-A and/or glucans. However, this bound and thereby neutralized LPS and/or lipid-A and/or glucan is of subordinate clinical importance. Of pathophysiologic importance is LPS, lipid-A or glucan (as e.g. the Candida-substance zymosan A), that is not-neutralized and/or free, the so-called reactive LPS or lipid-A or glucan, respectively, and can therefore react freely with susceptible cells as e.g. the monocytes of the blood. Releasing this neutralized LPS and/or lipid-A and/or glucan e.g. by heat pretreatment would falsificate the test result and would allow only a limited conclusion about the concentration of the not-neutralized LPS and/or lipid-A and/or glucan.
Basing on this state of the art the task of the present invention was to find a simple procedure and a corresponding suitable test system that allows to determine rapidly the content of active LPS and/or active lipid-A and/or active glucan of a sample, i.e. the LPS-reactivity and/or lipid-A reactivity and/or glucan reactivity.
A further task of the present invention was to find a procedure and a suitable test system that allows to differentiate active LPS or active lipid-A, respectively, from active glucan of a sample.
A further task of the present invention was to find a procedure and a suitable test system that allows to determine the active and/or free LPS and/or lipid-A and/or glucan of a sample and that is insensitive to neutralized LPS and/or lipid-A and/or glucan.
A still further task of the present invention was to find a procedure and a suitable test system operating with a linear kinetic and/or that is routine suitable.
A still further task of the present invention was to find a suitable test system that allows to omit the prior dilution and/or heat treatment of the sample to determine LPS and/or lipid-A and/or glucan.
Surprisingly it showed up that by addition of a suitable oxidant, if appropriate together with a detergent, such as e.g. Triton-X100®, preferably in a final concentration of up to 0,1%, and/or a solution mediator, as e.g. polyethylene glycol, preferably in a final concentration of up to 10%, the interference of the matrix of the sample (=surrounding of the parameter to be tested for) the disturbing factors on the Limulus-system could be eliminated.
Although it is known from EP-A-297,597, that by oxidative treatment of biological liquids the activity of serine proteases or of serine protease inhibitors can be determined, however this document does not refers to the usage of oxidants in the Limulus-assay. Additionly, according to the present invention it turned out that for the usage with the Limulus-system substantially higher oxidant doses are necessary that are e.g. 5–10 fold over the dose that are necessary to inactivate oxidant-sensible serine protease inhibitors.